Talk:20.109(S11):Initiate cell culture (Day2)

Plan for Day 2

Group 1 should arrive by 1:05 pm at the latest and immediately go to the tissue culture room. When everyone has arrived, we will thaw your cells in the water bath. After you have finished your culture preparations (ideally by 3 pm), you can take a 10 minute break to refresh your minds, and then will take a short quiz.

Group 2 should arrive by 2:45 pm at the latest and will begin by taking the quiz. If all goes well, you will begin working in the tissue culture room at 3 pm. If your culture preparations that involve a special physical set-up, you can come earlier and work in the extra tissue culture hood.

Notebooks may be handed in by Friday the 15th at 5 pm for the T/R section, and by Wednesday the 20th at 5 pm for the W/F section. (Earlier is certainly welcome for W/F folks if you are not away for the holiday.)

T/R

Arrival time (at latest!)	1:05 pm	2:45 pm
Team colour 1	Pink	Green
Team colour 2	Purple
Team colour 3	Red
Team colour 4	Yellow

W/F

Arrival time (at latest!)	1:05 pm	2:45 pm
Team colour 1	Purple	Red
Team colour 2	Blue	Green
Team colour 3	Orange
Team colour 4	Pink

Designs (T/R)

Team	Design
Yellow	We decided to test the effects of stiffness, through the use of low and high calcium concentrations, on the rate of dedifferentiation of chondrocytes. We expect there to be less dedifferentiation on stiffer alginate (high calcium concentration). Thus, we expect that the cells on a stiffer alginate will maintain a chondrocyte-like phenotype better than those on a less stiff surface, because similar results have been recorded using a smaller range of calcium concentration (Genes et al. 2003). We also plan to compare our results using medium viscosity alginate with those of Red and Orange, who are using low viscosity alginate.
Red	We predict the alginate in 200mM calcium will maintain chondrocyte phenotype better than alginate in 20mM calcium because the 200mM calcium forms a stiffer porous scaffold, allowing for the cells to form a better matrix
Orange	We decided to compare the cross-linking produced by high and low Calcium concentrations (20mM, 200mM) with the Alginate density and viscosity to see how each of these affects stiffness and resulting phenotypes of tissues. We used the 1% low viscosity alginate, and we wish to compare this with the same Calcium parameters tested in 2% low viscosity by the Red Group. In particular We would like to observe effects of increased cross-linking in low density alginate, versus less cross-linking in a higher percent alginate. density was shown to increase retention of certain mechanotranducers in the literature, and stiffness influences dedifferentiation to fibroblast-like so we would like to see if cross linking produces a comparable stiffness to effect collagen I expression in chondrocytes as 2% alginate at constant viscosities. Then conpare this to the effects of varying viscosity in 2% alginate (from Yellow and orange data). By varying three parameters it may be possibly to quantify expression of the two collagen types as well as characterize morphological differences for each set of conditions.
Blue	We will be preparing alginate cell scaffolds supplemented with none, low (0.05 mg/ml final), or high (0.5 mg/ml final) levels of hyaluronic acid (HA) in the presence (1 mg/ml final) and absence of exogenous collagen II. We will be examining the differentiation of stem cells into chondrocytes under these conditions by assaying collagen II and collagen I mRNA transcript levels (markers of cell type). We expect the cells under conditions of high HA and no exogenous collagen II to exhibit more of a fibroblastic phenotype, because of the results of Yoon et al. (2009). On the other hand, we expect cells under conditions of exogenous collagen II and low HA to exhibit more of a chondrocytic phenotype, because of the results of Bosnakovski et al. (2005). The interesting case will be the scenario with high HA and exogenous collagen II. We expect this case to yield some intermediate level of differentiation.
Purple	Our group collaborated with Blue and Green. The level of hyaluronic acid (HA) that we tested were 0.05mg/ml final, either with exogenous collagen II (1 mg/ml final) or without it. We expect the cells with no exogenous collagen II to show more fibroblast expression from Yoon et al (2009). The cells with the 0.05 mg/ml final concentration of HA we expect to exibit more chodrocytic phenotype from Bosnakovski et al. (2005). For the high HA and exogenous collagen II cells, an intermediate differentiation level is expected. (Please see blue group description for more details)
Pink	We are looking at the differences in the differentiation of chondrocytes into fibrocytes in the presence and absence of ascorbate. Both samples have 0.4 uL Collagen 2 and 1.6 uL alginate (1%, low viscosity). The control sample is chondrocytes without collagen 2.

Designs (W/F)

Team	Design
Yellow	We are testing the effects of extra glycine in the amount of collagen production. Since glycine is one of the main components of collagen (repeated Gly-Pro-Hyp sequences), we expect to see increased production of collagen just based on the availability of the reagents. We are particularly interested in the relative ratios of Type I and Type II collagen, and want to see if this ratio is preserved or distorted compared to a control sample with no glycine. We are testing this with 1.5% low-viscosity alginate and 800 uM glycine (double the amount of proline in the media) added to chondrocytes. We originally planned to study this effect in MSCs but had to change to chondrocytes due to availability of cells.
Red	We are looking at the effects of alginate stiffness by changing the amount of cross linking through low and high calcium chloride concentrations (20 mM and 200 mM respectively). We hypothesize that a higher calcium chloride concentration will result in more cross linking and thus stiffer alginate, causing less chondrocyte dedifferentiation. We are using 1% medium viscosity alginate and comparing our results with other groups using a different percentage and viscosity of alginate.
Orange	We will studying the effects of BMP-6 on chondrocyte differentiation. The control will have regular stem cell media, and the experiment will have 20 ng/mL BMP-6. Current literature contests if BMP-6 stimulates faster differentiation, so we wanted to see what our results could add to the debate.
Blue	Like the WF pink group, we are studying the effects of alginate viscosity on both the rate and extent of chondrocyte differentiation. Cells were prepared at a density of 5 million cells/mL for each sample with low and medium viscosity alginate (1.5% each). Like the pink group, we also believe that the lower viscocity alginate will allow for a more rapid chondrogenesis and will maintain the chondrocyte phenotype better than the medium viscosity alginate.
Purple	We are examining the effects of cross linking of 2% alginate on chondrocyte dedifferentiation. We are comparing the cross linking effects of 20mM and 200mM CaCl2 and expect with increasing calcium concentration, there will be decreased chodrocyte dedifferentiation; these results would reflect the outcome of the Genes et al. 2003 paper. This set up is similar to the Red, Orange, and Yellow groups from TR and will be used to compare reproducibility of results as well as changes in other variables (ie. 1% alginate, and other viscosities).
Green	In 2006, Bosnakovski et al. found that collagen type II can induce chondrogenesis in stem cells and maintain the chondrocyte phenotype in differentiated cells. In April 2011, Chang et al. found that a 2:1 ratio of chondrocytes:stem cells can promote collagen expression in the ECM. Since we are using collagen levels to measure the presence of chondrocytes, this is a strong indication that a 2:1 ratio of chondrocytes:stem cells can promote chondrogenesis. However, Chang et al. performed their experiments in the absence of a scaffold. In our experiment, we want to see if a 2:1 ratio of chondrocytes:stem cells can promote chondrogenesis in a 1.5% low-viscosity alginate scaffold as well. We will compare our 2:1 1.5% alginate case to both a stem cell-only case, and a chondrocyte-only case.
PINK	We prepared cultures at a cell density of 5e6 cells total. One was in 1.5% low viscosity alginate, one was in 1.5% medium viscosity alginate. We are looking for differences in bead viability based on alginate viscosity. We expect the low viscosity alginate will grow better and maintain chondrocyte phenotypes better than the medium viscosity alginate.